Inorganic nitrogen acquired by plants is ultimately converted to ammonium before being assimilated in organic nitrogen metabolism. One enzyme postulated to be involved in the assimilatory process is glutamate dehydrogenase (GDH), a group of ubiquitous enzymes found to be present in almost all organisms from microbes to higher plants and animals (Srivastava, H. S., R. P. Singh [1987] Phytochem. 26:597-610). GDH catalyses the reversible conversion of .alpha.-ketoglutarate to glutamate via a reductive almnation that utilizes reduced .beta.-nicotinamide adenine dinucleotide (NADH) or reduced .beta.-nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. The role of plant GDHs in the assimilation of ammonium into amino acids has been questioned since the discovery of the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway that is believed to be the favored pathway for ammonium assimilation in higher plants (Miflin, B. J., P. J. Lea [1976] Phytochem. 15:873-885).
The primary objection to GDH playing a major role in plant nitrogen metabolism is its low afiniy for ammonium that would require high intracellular ammnonium concentrations to function anabolically. Early evidence indicated that GDH is a catabolic enzyme catalyzing the deamination of glutamate with only a partially anabolic function in synthesizing glutamate (Wallgrove, J. C., N. P. Hall, A. C. Kendall, [1987] Plant Physiol. 83:155-158). The physiological role of large amounts of GDH present in various plant tissues and organelles is still unclear, and possible conditions under which GDH may play a significant role in carbon and nitrogen metabolism have not been resolved.
The majority of plant GDHs characterized to date are localized in the mitochondria; however, a GDH species differing in several properties (i.e., cofactor specificity, K.sub.m values, organelle localization, thermal stability) has been characterized from the chloroplast of a unicellular green alga Chlorella sorokiniana. C. sorokiniana cells have been shown to possess a constitutive, mitochondrial, tetrameric NAD-specific GDH (Meredith, M. J., R. M. Gronostajski, R. R. Schmidt [1978] Plant Physiol. 61:967-974), and seven ammonium-inducible, chloroplast-localized, homo- and heterohexameric NADP-GDH isoenzymes (Prunkard, D. E., N. F. Bascomb, R. W. Robinson, R. R. Schmidt [1986] Plant Physiol. 81:349-355; Bascomb, N. E., R. R. Schmidt [1987] Plant Physiol. 83:75-84). The seven chloroplastic NADP-GDH isoenzymes were shown to have different electrophoretic mobilities during native-PAGE, and presumably result from the formation of homo- and heterohexamers composed of varying ratios of .alpha.- and .beta.-subunits (53.5 and 52.3 kilodaltons, respectively).
Chlorefla cells cultured in 1 to 2 mM ammonium medium accumulate only the .alpha.-homohexamer (Bascomb and Schmidt, supra). The addition of higher ammonium concentrations (3.4 to 29 mM) to nitrate-cultured cells results in the accumulation of both .alpha.- and .beta.-subunits in NADP-GDH holoenzymes (Prunkard et al., supra; Bascomb and Schmidt, supra; Bascomb, N. F., D. E. Prunkard, R. R. Schmidt [1987] Plant Physiol. 83:85-91). Prunkard et al. (Prunkard, D. E., N. F. Bascomb, N F, W. T. Molin, R. R. Schmidt [1986] Plant Physiol. 81:413-422) demonstrated that the NADP-GDH subunit ratio and isoenzyme pattern is influenced by both the carbon and nitrogen source as well as the lift conditions under which cells are cultured.
.alpha.- and .beta.-NADP-GDH homohexamers purified from Chlorella cells have strikingly different ammonium K.sub.m values; however, the K.sub.m values for their other substrates are very similar. The .alpha.-homohexamer (composed of six identical .alpha.-subunits) that catalyzes the biosynthesis of glutamate is allosterically regulated by NADPH and possesses an unusually low K.sub.m for ammonium that ranges from 0.02 to 3.5 mM, depending on the NADPH concentration (Bascomb and Schmidt, supra). The K.sub.m value for ammonium of the .alpha.-homohexamer is the lowest reported ammonium K.sub.m for any plant GDH characterized to date. In contrast, the .beta.-homohexamer (catabolic form) is a non-allosteric enzyme with an ammonium K.sub.m of approximately 75 mM. From these studies involving purified enzymes, it is postulated that the heterohexamers have varying degrees of affinity for ammonium. However, no kinetic analyses have been performed on purified heterohexamers.
Although the .alpha.- and .beta.-subunits have distinct in vivo turnover rates (Bascomb et al., supra) and the corresponding homohexamers have remarkably different ammonium K.sub.m values, the .alpha.- and .beta.-subunits are derived from precursor proteins of nearly identical size (ca 58,000 Daltons) and were shown to have very similar peptide maps (Prunkard et al., supra; Bascomb and Schmidt, supra). Moreover, polyclonal antibodies prepared against the .beta.-homohexamer are capable of immunoprecipitating all of the NADP-GDH isoenzyes (Yeung, A. T., K. J. Turner, N. F. Bascomb, R. R. Schmidt [1981] Anal. Biochem. 10:216-228; Bascomb et al., supra), but do not crossreact with the mitochondrial NAD-GDH. In addition, previous research in this laboratory provided genomic cloning and southern blot evidence that indicated the C. sorokiniana genome possesses a single NADP-GDH structural gene (Cock, J. M., K. D. Kim, P. W. Miller, R. G. Hutson, R. R. Schmidt [1991] Plant Mol. Biol. 17:17-27).
The C. sorokiniana nuclear-encoded chloroplastic NADP-specific glutamate dehydrogenases are the only chloroplastic localized GDH sequences isolated and characterized from plants. Although the Chlorella GDH isoenzmes had been well characterized, it has been discovered in the present invention that the two mature subunits arise via specifc processing of two similar precursor proteins encoded by two mRNAs formed by alternative splicing of a pre-mRNA derived from a single nuclear gene. Furthermore, the identification of the cleavage site and amino-terminal peptide sequence of the mature functional GDH subunits critical to understanding the enzymatic regulation previously demonstrated in vitro had not been accomplished prior to the present invention.